The invention is related to molecular methods for the diagnostic detection of neoplasms. It is particularly related to the identification of neoplastic cells and the characterization of tumors by determination of their genotypic properties.
The somatic mutational theory of cancer (Knudson, 1971; 1985) has gained overwhelming support from the finding that mutations activate the malignant potential of oncogenes and inactivate the repressor function of tumor suppressor genes (Bishop, 1991). However, the etiology of these mutations remains poorly understood. There is no agreement on the estimation of the relative contribution by genotoxic agents and of spontaneous errors during DNA replication and repair to the genesis of these mutations in cancer. The importance of DNA replication and repair in malignant transformation is demonstrated by the association of various biochemical defects in these processes with hereditary diseases that predispose to cancer. The genes encoding protein factors involved in the DNA synthesis and repair pathways are beginning to be isolated and characterized. A causal link between defective factors involved in DNA metabolism and the origin of mutations in oncogenes and tumor suppressor genes is not yet in sight. The concept that spontaneous errors in the replication process may be fundamental in transformation was put forward in an attempt to explain the increased chromosomal alterations of cancer cells at late stages of tumor progression (Loeb et al., 1974). A defect in an enzyme involved in DNA replication could generate an enhanced rate of errors in the tumor cell variants continuously selected as tumor progression takes place (Foulds, 1954; Nowell, 1976). This defect could be therefore causative of the apparent genetic instability of malignant cells (Schimke et al., 1986). However, a critical prediction of this hypothesis, an increased mutation rate in tumor cells, has not been conclusively demonstrated despite intensive efforts (Harris, 1991; Loeb, 1991).
Hereditary diseases that predispose to cancer have also been associated with various biochemical defects in the processes of DNA replication and repair (Lindahl et al., 1991), implying that mutations in the genes encoding the protein factors involved in the DNA synthesis and error repair pathways could lead to malignant transformation. Again, however, no causal link between defective factors in DNA metabolism and the emergence of mutations in oncogenes has been established.
Colorectal cancer is one of the best characterized examples of the multistage nature of neoplastic development. A dominant oncogene, c-K-ras (Bos et al., 1987; Forrester et al., 1987) and at least three distinct tumor suppressor genes: p53 (Baker et al., 1989), DCC (Fearon et al., 1990), and MCC/APC (Kinzler et al., 1991a; 1991b; Groden et al., 1991) are consistently involved in colorectal tumorigenesis (Fearon and Vogelstein, 1990). In addition to the genetic alterations in these critical genes, other apparently random alterations in genome structure are observed in tumors of the colon and rectum, which exhibit remarkably heterogenous distribution in the extent of this genetic damage at the chromosomal level (Vogelstein et al., 1988; 1989).
Mutations corresponding to genetic alterations in tumor cells can comprise loss of heterozygosity (LOH) resulting from the loss of chromosomal sequences accompanying the inactivation of tumor suppressor genes, as well as increases in chromosomal sequences expressed in the aneuploidy of the cancer cell observed cytogenetically. In colorectal cancer, about 20% of the genomic sequences undergo losses of heterozygosity.
The search for genetic alterations that can be used for the detection of mutations associated with neoplastic change, either as deleted or amplified sequences, is complicated by the analytical requirements for analyzing closely related genetic sequences, the polymorphism among individuals, and the small amount of genetic material available for analysis.
DNA fingerprinting, based on polyacrylamide gel electrophoresis of labeled DNA restriction fragments, is a powerful technique for the comparative analysis of closely related genomes. It has been applied to the detection of polymorphisms during malignant transformation (Thein et al., 1987; de Jong et al., 1988), and to studies of the clonality of tumors, both primary and metastatic (Smit et al., 1988; Fey et al., 1988). DNA fingerprinting has been enhanced by its use in combination with the polymerase chain reaction (PCR) (Saiki et al., 1985; Mullis et al., 1987), which allows the reproducible amplification of primer-defined DNA sequences. A PCR-based DNA fingerprinting technique, called arbitrarily primed PCR, or AP-PCR (Welsh et al., 1990), utilizes amplification with a single arbitrary primer. The first cycles of amplification are performed at a low annealing temperature, which is raised in subsequent cycles to increase stringency. In the initial stages, the primer hybridizes to many sequences in the total genomic DNA. When the temperature is increased, only the best matches of the initial annealing events are amplified further, generating a number of discrete bands that provide a fingerprint of the cell genome. This approach has been used for mapping DNA polymorphisms in various prokaryotic and eukaryotic systems (Welsh et al., 1990; 1991). In a recent study, AP-PCR detected tumor-specific somatic genetic alterations by comparison of the fingerprints from normal and tumor tissue of the same individual. Cloning and analysis of the altered DNA bands from tumors of the colon and rectum (Peinado et al., 1992) determined that somatic deletions of a few nucleotides had occurred in a subset of colon tumors.
It is therefore an object of the invention to provide methods based on DNA fingerprinting and auxiliary amplification techniques, such as PCR, that can be used to identify genetic alterations that are predictive or diagnostic of neoplastic change in cells.
It is another object of the invention to provide a method for the diagnostic identification of a particular class of tumors based on the presence of these mutations.
It is yet another object of the invention to provide a method for the molecular diagnosis of tumors that have predictive value of malignant transformation.
It is yet another object of the invention to provide a simple diagnostic method for the molecular diagnosis of tumors that have predictive value for cancer prognosis.
The invention provides a method for identifying tumors comprising the steps of obtaining tissue or sample DNA from a potential tumor, and analyzing DNA or RNA of the sample from at least one genomic region typically containing reiterated sequences to determine whether insertion or deletion mutations have occurred in the reiterated sequences.
In a preferred embodiment, the method of the present invention utilizes amplification by PCR of tumor tissue DNA. According to preferred protocols, the PCR amplification procedures are carried out so that at least two different genomic regions containing reiterated sequences are amplified.
In these protocols, the amplification includes the step of contacting the sample DNA with a first set of primers adapted to amplify a first region thereof containing reiterated sequences, and contacting the sample DNA with a second pair of primers adapted to amplify a second region thereof containing reiterated sequences. In another embodiment, the method of amplification is arbitrarily-primed PCR amplification. In an alternative embodiment, the amplification is ligase chain reaction amplification.
In a particularly preferred embodiment of the invention, amplification only occurs if insertions or deletions in the region have occurred. In preferred protocols related to this aspect of the invention also, the amplification step can comprise contacting the DNA with a first set of primers complementary to sequences flanking the reiterated sequences, amplification of the region occurring only if a first number of insertions or deletions has occurred, and a second set of primers complementary to sequences flanking the reiterated sequences, amplification of the region occurring only if a different, second number of insertions or deletions has occurred. The preferred protocol the amplification step can comprise contacting the DNA with a third set of primers complementary to sequences flanking the reiterated sequences, amplification of the region occurring only if a third number of insertions or deletions has occurred, wherein the third number is different from the first and second numbers.
In an alternative embodiment of the invention, there is provided a method for identifying tumor cells comprising the steps of (a) obtaining a sample containing DNA from a potential tumor, (b) contacting the sample with primers that are complementary to a predetermined portion of the DNA which typically contains reiterated sequences, (c) subjecting the sample and the primers to amplification steps to amplify such DNA, and (d) determining whether amplification of DNA occurs, wherein amplification indicates the presence of tumor cells. In a preferred embodiment, the method further comprises the steps of performing a control assay by also obtaining control sample containing DNA from normal tissue, subjecting the control sample to steps (b) through (d), above. In a particularly preferred embodiment, the control sample and the tumor sample are obtained from a common patient. In a most preferred embodiment of the invention, amplification occurs only when insertions or deletions have occurred.
According to another particularly preferred embodiment, the test for inserted or deleted sequences is carried out by the Southern hybridization method.
The invention also provides a diagnostic kit to identify tumor cells, comprising a compartmented enclosure containing at least one set of primer pairs complementary to a predetermined portion of DNA from the tumor cells which ordinarily contain reiterated sequences, and reagents to carry out PCR amplification, and optionally, control DNA sequence samples, a polyacrylamide gel slab, and electrophoresis buffer. The invention also includes a diagnostic kit adapted to identify insertion or deletion-associated neoplasms by use of the ligase chain reaction (LCR) process comprising a first pair of ligase chain reaction primers complementary to a predetermined region of DNA which ordinarily contains reiterated sequences, amplification occurring only if one or more insertions or deletions have occurred in a reiterated sequence contained within said region; and reagents for performing ligase chain reaction using said primers. In a preferred embodiment of this aspect of the invention, the kit further comprises a second pair of primers adapted to amplify said region only if a different, second number of insertions or deletions have occurred in the reiterated sequence. In yet another preferred embodiment of this aspect of the invention, the kit further comprises a third pair of primers adapted to amplify said region only if a third number of insertions or deletions have occurred in said reiterated sequence, wherein the third number is different from the first and second numbers.